Our primary approach to develop an effective prophylactic vaccine against HIV utilizes a novel immunogen called the Full Length Single Chain (FLSC) that consists of gp120 derived from HIV-1 genetically linked via a 20 amino acid linker to the D1D2 domains of human CD4. When rhesus macaques were inoculated with versions of FLSC that contain CD4 derived from rhesus macaques and either HIV gp120 or SIV gp130, significant protection against rectal challenge with multiple, low doses of either an R5 tropic, heterologous SHIV162P3, or heterologous SIVmac251 was observed. These observations propelled FLSC into preclinical development and evaluation in a phase 1 clinical trial (supported by BMGF, MHRP, NIAID). Further, vaccination with DNA expressing FLSC and IL-12 administered by electroporation in macaques induces multifunctional T cells that are known to correlate with protection and improved efficacy. In our most recent macaque study, a FLSC DNA prime / protein boost regimen yielded 75% efficacy against a cross clade challenge. In collaboration with others, we have also developed a DNA / protein co-delivery regimen that evokes higher-titered, longer-lived anti-FLSC responses. However, we believe that our approach can be dramatically improved by targeting these anti-FLSC responses to mucosal sites where HIV enters the host and establishes infections. To improve mucosal immune responses we sought to develop a mucosal homing DNA adjuvant that targets lymphocytes to mucosal immune effector sites after systemic vaccination. In preliminary studies, we identified two putative mucosal homing DNA adjuvants. Under phase I, we demonstrated that one of these putative adjuvants does indeed induce the homing of lymphocytes to mucosal immune effector sites after systemic i.m. immunization. Under phase II, we intend to expand upon these observations and demonstrate that this adjuvant targets vaccine-specific immune responses to mucosal sites in primates.